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High-Throughput Sequencing Techniques for the Detection and Survey of Marine Non-Indigenous Species : a Valuable Addition to Traditional Methods ? Marjorie Couton

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High-Throughput Sequencing Techniques for the Detection and Survey of Marine Non-Indigenous Species : a Valuable Addition to Traditional Methods ? Marjorie Couton High-throughput sequencing techniques for the detection and survey of marine non-indigenous species : a valuable addition to traditional methods ? Marjorie Couton To cite this version: Marjorie Couton. High-throughput sequencing techniques for the detection and survey of marine non- indigenous species : a valuable addition to traditional methods ?. Ecosystems. Sorbonne Université, 2020. English. NNT : 2020SORUS039. tel-03180937 HAL Id: tel-03180937 https://tel.archives-ouvertes.fr/tel-03180937 Submitted on 25 Mar 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Sorbonne Université Ecole doctorale 227 Sciences de la nature et de l’Homme : évolution et écologie UMR7144 Adaptation et Diversité en Milieu Marin Equipe Dynamique de la Diversité marine High-throughput sequencing techniques for the detection and survey of marine non-indigenous species: a valuable addition to traditional methods? Le séquençage haut-débit pour détecter et étudier les espèces marines non- indigènes : un complément pertinent aux méthodes traditionnelles ? Par Marjorie Couton Thèse de doctorat en Ecologie Marine Sous la direction de Frédérique Viard et Thierry Comtet Présentée et soutenue publiquement le 29 octobre 2020 Devant un jury composé de : Pr TURON Xavier, Professeur, Centre d’études avancées de Blanes Rapporteur Dr RODRIGUEZ-EZPELETA Naiara, Directrice de recherche, AZTI Rapporteure Dr NUNES Flavia, Directrice de recherche, Ifremer Examinatrice Pr DESTOMBES Christophe, Professeur, Sorbonne Université Examinateur Dr VIARD Frédérique, Directrice de recherche, CNRS Directrice de thèse Dr COMTET Thierry, Chargé de recherche, CNRS Co-directeur de thèse Remerciements En tout premier lieu, j’aimerais remercier mes superviseurs, Frédérique Viard et Thierry Comtet pour leur soutien et leurs encouragements. Toutes nos discussions, formelles et informelles, ainsi que leurs conseils et remarques avisés, m’ont permis de ne pas me reposer sur mes acquis et de toujours me remettre en question. Tous les progrès réalisés et la maturité acquise lors de ces trois dernières années n’auraient pas été possibles sans vous. Bien plus qu’un support scientifique, vous m’avez permis de réaliser cette thèse dans une ambiance bienveillante et amicale, ce qui représente, à mon sens, la majeure partie d’un travail réussi. Contrairement à ce qui pourrait transparaitre au travers d’un manuscrit, une thèse est une aventure collective et aucun des résultats décrits ici n’auraient pu être acquis sans l’aide d’un grand nombre de personnes. C’est le cas de Claire qui m’a épaulé tout au long de ce périple, de l’échantillonnage jusqu’au séquençage. Ses compétences et son expérience au laboratoire m’ont évité pas mal de faux pas. C’est également le cas de Laurent qui a réalisé toutes les identifications morphologiques présentées et dont l’expérience du terrain a été d’une aide précieuse. Enfin Erwan m’a également accordé de son temps pour jeter un œil avisé de bioinformaticien sur mes travaux et me conseiller dans mes analyses. A vous trois un, grand merci. J’aimerais également rendre hommage à tous ceux qui m’ont aidée pour les échantillonnages. Merci aux plongeurs, Mathieu, Wilfried et Yann, pour leur indéfectible bonne humeur, merci aux marins, François, Gilles et Noël pour leur gentillesse, malgré des conditions parfois difficiles, et merci également à Stéphane et Jérôme pour patience et leur efficacité. Je suis aussi reconnaissante envers Marion et Aurélien pour leur aide au laboratoire, envers l’équipe du CRBM et notamment Gaëtan pour la mise en place de matériel pour la remontée des panels de recrutement, ainsi qu’à la plateforme Genomer, et surtout Gwenn et Erwan, pour leur aide lors des séquençages. Enfin, parce que les interactions sociales sont très importantes pour un travail efficace (cf. le confinement), je tiens à remercier tous ceux avec qui j’ai pu passer des moments de discussions informelles et de détente pendant ces trois ans. Merci à toute l’équipe ABiMS pour leur accueil chaleureux, leurs pauses café/gâteaux de 10h30, les blagues du mercredi et surtout pour ne pas m’avoir retiré de l’alias igm3 pour que je puisse continuer à venir m’incruster de temps en temps après avoir déménagé dans un autre bâtiment. Merci à Delphine pour nos randonnées, parfois un peu plus longues que prévues à cause de notre mauvais sens de l’orientation, merci à Anaëlle et Anaïs pour les discussions autour d’un café et pour m’avoir supportée (dans les deux sens du terme) lors de la dernière ligne droite, et merci à Romain pour nos balades quotidiennes qui m’ont permis de me changer un peu les idées pendant la rédaction de ce manuscrit. Et parce que travailler pendant le confinement n’a pas été une mince affaire, merci à mes sœurs, Gaëlle et Solène, pour m’avoir permis de conserver ma santé mentale pendant ces deux mois compliqués. Et pour tous ceux que j’ai pu oubliés et qui ont participé, de près ou de loin, à la création de ce travail, MERCI ! Table of contents Introduction 1 1. Biological invasions: one facet of the global change …………………….……………………..3 a. Biological introductions – definition and global overview ..………..…….………………………..…..3 b. A dynamic process (from introduction to invasion, and failure) .……….………………….…..……5 c. Consequences and management of biological invasions ..……………………………..…………..….7 2. Some important properties of marine biological introductions ………….………………..10 a. Introduction vectors responsible for specific patterns and processes ……………….………...10 b. Marinas and ports as invasion hubs ………………..…………………………………….……….…………….13 3. Molecular tools for the biomonitoring of non-indigenous species ………………………16 a. A furnished DNA-based toolbox with many applications …………………..……………….……….17 b. NIS identification through DNA barcoding: general principle .……..………………….…………..20 c. Producing and processing metabarcoding data .………………………..………………….…………….22 Thesis objectives .…………………………………………………...………………..………………………………..32 Chapter I : High-Throughput Sequencing on preservative ethanol is effective at jointly examining infra-specific and taxonomic diversity, although bioinformatics pipelines do not perform equally 37 Preamble ……………………………………………………………………………………………………………..……39 Abstract …………………………………………………………………………………………………………………….41 Introduction ……………………………………………………………………………………………………………...42 Materials and methods ………………….…………………………………………………………………………..43 a. Case study & sampling …………..……………………………………………..………………………………...……43 b. Sanger sequencing on individual zooid (SSIZ) ………………………..………..…………………………..45 c. High-throughput sequencing on assemblages (HTSA) ……………………..……..………………..…45 Sample processing ……………………………………………………….................…………………...……45 Reads processing …………………………………………..………………………….………………………...47 d. Data analyses ……………………………………..…………………………………………………………………………48 Assignment ………………………………………………………………………………………………………....48 Haplotype comparison ………………………………………………………………………………….…….48 Diversity indices ……………………………………………………………………………………………..……48 Results ……………………………………………………………………………………………………………………….49 a. Sanger sequencing on individual zooid (SSIZ) ...…………………………………………………………...49 b. Species assignment ………………………………………………………………………………………………………49 c. Pipeline performance for HTSA-based haplotype detection ………………………………………..50 d. Population diversity indices ………………………………………………………………………………………….54 Discussion ………………………………………………………………………………………………………………….55 a. Ethanol-based DNA is a valid non-destructive alternative to bulkDNA, even after several months of storage ………………………………………………………………………………………..…..55 b. Specific primers can improve the quantitative use of HTSA data …………………………………56 c. Careful choice of bioinformatics pipeline is needed to examine genetic diversity ………57 d. Improving haplotype detection – a matter of compromise ………………………………………….58 References …………………………………………………………………………………………………………………60 Chapter II : How effective is metabarcoding for studying communities living in marinas? 65 Preamble ………………………………………………………………………………………………….……………….67 Chapter II.1: Non-indigenous and native species from biofouling communities in marinas: the (un)detected fraction from environmental DNA metabarcoding ..........…67 Abstract ………………………………………………………………………………………………..……………………69 Introduction ……………………………..………………………………………………………………………………..69 Materials and methods ………………………………..…………………………………………………………….71 a. Sampling …………………………………………………………………………………………………..…………………..71 b. DNA extraction …………………………………………………………………………………………..…………………73 c. Library preparation and sequencing …………………………………………………………..………………...74 d. Reads processing and filtering ……………………………………………………………………..………………76 e. Taxonomic assignment ………………………………………………………………………………..……………….76 f. Comparison between quadrats and metabarcoding datasets ………………….………..………...77 Results .………………………………………………………………………………………………………………………78 a. Reads processing and taxonomic assignment …………………………………………….……………….78 b. Comparison of quadrats (morphology-based) and eDNA metabarcoding datasets .…..80 c. Effect of the sampling strategy on taxa detection ……………………………………………….……….84 Discussion .…………………………………………………………………………………………………………………86
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